Design, synthesis and biological evaluation of Pontin ATPase inhibitors through a molecular docking approach

Article abstract of DOI:10.1016/j.bmcl.2014.04.003

A virtual screening strategy, through molecular docking, for the elaboration of an electronic library of Pontin inhibitors has resulted in the identification of two original scaffolds. The chemical synthesis of four candidates allowed extensive biological evaluations for their anticancer activity. Two compounds displayed an effect on Pontin ATPase activity, and one of them also exhibited a noticeable effect on cell growth. Further biological studies revealed that the most active compound induced apoptotic cell death together with necrosis, this latter effect being likely related to the cellular balance of ATP regulation.

Full text of DOI:10.1016/j.bmcl.2014.04.003

Bioorganic & Medicinal Chemistry Letters 24 (2014) 2512–2516
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and biological evaluation of Pontin ATPase
inhibitors through a molecular docking approach
Judith Elkaim a, , Marc Lamblin a,1, , Michel Laguerre a, Jean Rosenbaum b, Patrick Lestienne b, Laure Eloy c,
Thierry Cresteil c, François-Xavier Felpin d, , Jean Dessolin a,
⇑
⇑
a Université de Bordeaux, CNRS UMR 5248, CBMN, IECB, 2 rue Robert Escarpit, 33607 Pessac, France
b Physiopathologie du Cancer du Foie, INSERM U1053-Université de Bordeaux, 146 rue Léo Saignat, F-33076 Bordeaux Cedex, France
c Institut de Chimie des Substances Naturelles, UPR 2301, CNRS, Avenue de la Terrasse, 91198 Gif-sur-Yvette, France
d Université de Nantes, CNRS UMR 6230, CEISAM, UFR des Sciences et des Techniques, 2 rue de la Houssinière, 44322 Nantes Cedex 3, France
a r t i c l e i n f o
a b s t r a c t
Article history:
A virtual screening strategy, through molecular docking, for the elaboration of an electronic library of
Pontin inhibitors has resulted in the identification of two original scaffolds. The chemical synthesis of
four candidates allowed extensive biological evaluations for their anticancer activity. Two compounds
displayed an effect on Pontin ATPase activity, and one of them also exhibited a noticeable effect on cell
growth. Further biological studies revealed that the most active compound induced apoptotic cell death
together with necrosis, this latter effect being likely related to the cellular balance of ATP regulation.
Ó 2014 Elsevier Ltd. All rights reserved.
Received 10 March 2014
Revised 31 March 2014
Accepted 2 April 2014
Available online 13 April 2014
Keywords:
ATPase activity
Pontin
Molecular docking
Small-molecule inhibitor
Enzymatic assay
Pontin and Reptin are ATPases that belong to the AAA+ (ATPases
associated with diverse cellular activities) family.1 As such, these
proteins are implicated in multiple and very different cellular func-
tions,2 as reflected by the variety of names they are called, such as
RuvBL1 (RuvB-like),3 Rvb1,4 TAP54a (TIP60 associated protein)5 or
TIP49 (TATA-box binding protein)6–8 for Pontin, and RuvBL2, Rvb2,
TAP54b or TIP489 for Reptin.10 They contain characteristic Walker
A and B domains, essential respectively for the binding and the
hydrolysis of ATP and structural studies have highlighted their
ability to form ring-shaped oligomeric complexes.1,11,12 The crys-
tallographic structure of Pontin alone was resolved as an hexamer
in 2006,13 and in 2011, a dodecameric assembly composed of two
heterohexamers of alternating units of Pontin and Reptin has been
published by the same team.14 The structures display bound ADP
for Pontin alone, or a mixture of bound ATP and ADP for the dode-
camer of Pontin and Reptin. A similar heterododecameric structure
has been reported by another group using cryo-electron micros-
copy.15 The ATP/ADP binding site, which is located at the interface
of two subunits, seems to involve residues from both chains.13,14
Recently, several reports have hinted at a link between Pontin
and Reptin and various types of cancer.16 We notably found that
both proteins were overexpressed in human hepatocellular carci-
noma where they are required for cell viability and prolifera-
tion.17,18 They also interact with oncogenic transcription factors
such as beta-catenin or c-Myc.19,9 Finally, they are also required
for the assembly and function of telomerase20 and for the stability
and function of mTOR.21,22 As suggested by the use of mutants
devoid of ATPase activity, most functions of these proteins require
an ATPase activity, including those implied in cancer,9,20–25 indicat-
ing that the inhibition of the ATPase activity of Pontin and/or
Reptin could be of special interest for cancer therapy. In a previous
Letter,26 we have disclosed the discovery of the first four small
molecules able to inhibit the ATPase activity of Pontin. This was
achieved through the combination of molecular docking of com-
pounds from commercially available libraries into the ATP binding
site and in vitro ATPase assays. Further testing showed that one of
these four molecules was competitive with ATP. Encouraged by
these promising results we decided to look for Pontin inhibitors
among original molecules from our laboratories. At this time,
4-hydroxy-2-pyridone and 4-hydroxy-2-quinolone moieties were
of special interest to us because of their apparent similitude with
quinones and napthoquinones. Starting from these scaffolds,
simple chemical transformations were envisioned to introduce
chemical diversity in an easy manner and prepare a set of innovative
⇑
Corresponding authors. Tel.: +33 5 4000 3029; fax: +33 5 4000 2200 (J.D.).
E-mail addresses: fx.felpin@univ-nantes.fr (F.-X. Felpin), j.dessolin@iecb.
u-bordeaux.fr (J. Dessolin).
1
Current address: OXELTIS; Cap Gamma, 1682 rue de la Valsière, 34189 Montpellier,
France.
Contributed equally to this work.
http://dx.doi.org/10.1016/j.bmcl.2014.04.003
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

J. Elkaim et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2512–2516
2513
NH
NH
NH2
NH2
H
N
H
N
OH
OH
O
O
N
H
O
O
N
H
O
O
2
1
NH
CO2H
CO2H
OH
N
H
OH
N
H
N
H
N
H
4
3
Figure 1. Selected structures from in silico studies to be prepared by chemical synthesis.
NH2
NO2
CO2Me
OBn
OBn
N
OBn
OBn
N
O
(b)
(a)
(c), (d)
(a), (b)
R
R
N
OBn
OBn
N
OBn
OBn
OBn
Cl
9
8
, 98%
6a
Pyr : , 73%
5a
5b
Pyr :
Qui :
CO2Bn
R
6b
Qui : , 79%
Boc
OBn
N
N
H
(e)
NH
HN
H
N
OBn
NH
CO2H
(c), (d)
O
OH
N
H2
10a, R = 3-indole : 52%
10b
N
OBn
3
, 53%
NH
NH3Cl
, R = Ph : 57%
H
N
Pyr : 7a, 70%
7b
N
H
O
O
OH
Qui : , 72%
CO2H
Cl
O
OH
N
H2
O
O
N
H
1, 63% (two steps)
4, 61%
NH
NH3Cl
N
H
H
N
OH
O
Scheme 2. Preparation of compounds 3–4. Reagents and conditions: (a) LiAlH4,
THF, 0–25 °C, 0.5 h; (b) stabilized 2-iodoxy benzoic acid, THF, 60 °C, 0.5 h; (c)
phenylalanine benzyl ester or -tryptophan benzyl ester, MS 4 Å, CH2Cl2, MeOH,
25 °C, 2 h; (d) NaBH4, À5 °C, 3 h; (e) H2, Pd/C, MeOH, 25 °C, 12 h, then HCl.
L-
N
H
2, 57% (two steps)
D
Scheme 1. Preparation of compounds 1–2. Reagents and conditions: (a) Fe, acetic
acid, reflux, 0.5 h; (b) Boc-( )-Trp-OH, EDC, HOBt, di-isopropyl ethyl amine, DMF,
25 °C, 12 h; (c) TFA, CH2Cl2, 0–25 °C, 0.5 h; (d) H2, Pd/C, MeOH, 25 °C, 12 h, then HCl.
L
modifications and implementations were proposed and introduced
on the original scaffolds. The changes were oriented either by the
experience of the organic chemists in the synthesis of this family
of compounds, or by the potential activity increase brought by
some functional groups, as suggested by previous virtual screen-
ing. If the envisioned chemical modifications were not judged fea-
sible, they were not incorporated. The final database contained
more than 800 original molecules designed for their easy access.
All docking calculations were conducted using Autodock Vina
1.0.2.29 The docking grid was a box with the following dimensions:
20 Â 22 Â 20 Å, in order to incorporate all residues of the cavity
with a margin of 3 Å in all directions. As already stated, 74 amino
acids from the first sub-unit, plus 6 from the second sub-unit were
included, at least partially, in the docking box. The whole receptor
was kept rigid during the docking, while all the ligands were fully
flexible. The poses obtained were rescored with DrugScore30,31 and
XScore32 then consensus scoring determined the most probable
ligands as already published.26 A short list of 15 compounds was
proposed to the chemists and 4 were immediately chosen to be
prepared, because of their synthetic accessibility.
Molecular docking of molecules from the virtual database led us
to the selection of several compounds, among which molecules
1–4, depicted in Figure 1, were the most easily accessible for the
campaign of chemical synthesis.
We recently described the preparation of 3-aryl-2,4-oxypyri-
dines and 3-aryl-2,4-oxyquinolines through a practical Pd/C-
catalyzed Suzuki–Miyaura reaction.33 Thereby, with the aid of this
efficient methodology, we prepared the 4-hydroxy-2-pyridone
(Pyr) and 4-hydroxy-2-quinolone (Qui) cores of our targets 1–4.
small compounds. Parallel to the organic chemistry effort, an in-
house virtual chemical database based on the chosen scaffolds
was simultaneously prepared, then extended along with the syn-
theses progress. This database contains all the compounds believed
to be accessible through synthetic chemistry.
The already published26 procedure was repeated in this study.
Briefly, the crystal structure of the hexamer of Pontin alone with
bound ADP was obtained from the Protein Data Bank, code
2C9O.13 Two adjacent subunits, forming a complete ATP/ADP bind-
ing site, were extracted from the ring-shaped hexamer. The result-
ing homodimer was used as the receptor for docking. It was formed
of one subunit as the main docking area, while the second sub-unit
was positioned on top of the cavity, as if to forbid access to the
exterior, as observed in the published crystal structure. Two miss-
ing loops in the crystal structure (residues 142–155 and 248–276)
were not completed since they were far enough from the nucleo-
tide binding site and did not impact the docking results. Water
molecules were removed from the receptor, then missing hydro-
gen atoms were added while applying Charmm Forcefield in Dis-
covery Studio 3.1 (Accelrys). The structure with bound ADP was
minimized while the backbone was fixed, using a Steepest Descent
algorithm (2000 step, gradient 0.01).
The virtual chemical database was prepared with Discovery
Studio 3.1. Two original moieties of special interest within
our group were chosen: pyridone27 and quinolone.28 Several

2514
J. Elkaim et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2512–2516
80
70
60
50
40
30
20
10
0
Compound 1
Compound 2
Compound 3
Compound 4
KB
MCF7
HCT116
HL60
Figure 2. Growth inhibition in human cancer cell lines.
100
90
80
70
60
50
40
30
20
10
0
Necrotic cells
Late apoptosis
Intact cells
Early apoptosis
24 h
48 h
Figure 3. Apoptosis in HL60 cells treated with compound 1 (percentage of cells).
Table 1
On the other hand, the preparation of compounds 3–4 started
from the 3-(4-methylbenzoate)-2,4-benzyloxyquinoline
Activation of caspase 3 in HL60 cells by compound 1
8
Caspase 3 activity
(Scheme 2). Conversion of the ester function into aldehyde was
realized through an efficient two step sequence giving a quantita-
tive yield of the expected compound 9. The reductive amination of
24 h
48 h
100 16
1251 18
119
131 12
247 87
169 29
HL60
Doxo 1 lM
100
178
116
111
134 15
306 22
7
6
5
7
9 with D-tryptophan benzyl ester and L-phenylalanine benzyl ester
1 5
lM
6
provided 10a and 10b in correct yields. Last, a Pd/C-catalyzed
hydrogenolysis of the benzyl protecting groups furnished the
expected targets 3 and 4.
1 10
1 20
1 50
l
l
l
M
M
M
As mentioned, our previous study revealed that 4 commercial
compounds were able to inhibit the ATPase activity of His-tagged
purified Pontin in the micromolar range concentration. Three
among these also inhibited cell tumor growth in vitro, indicating
that the enzymatic assay could be helpful for the identification of
novel inhibitors. The Malachite Green assays were conducted fol-
lowing the already published protocol.26 The ATPase activity of
The cleavage of DEVD was measured and expressed as a percentage of activity in
control cells.
For instance, compounds 1–2 were synthesized from the nitro-
derivatives 5a–b following the sequence depicted in Scheme 1.
Iron-mediated reduction of the nitro function led to the corre-
sponding anilines 6a–b in 73% and 79% yields respectively. Then,
the four synthesized molecules 1–4 were measured at a 100 lM
concentration. From this study, we noticed that compounds 1
and 2 reduced the inorganic phosphate (Pi) release in a dose
dependent manner. ATPase inhibitors 1 and 2 displayed an IC50
ligation of the latter, with N-Boc-L-tryptophan followed by protect-
ing groups cleavage furnished the targeted compounds 1–2 with
good overall yields.34
at 9 and 18 lM, respectively, and reduced the enzymatic activity

J. Elkaim et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2512–2516
2515
700
600
500
400
300
200
100
0
released LDH
total LDH
24 h
48 h
Figure 4. Total LDH and LDH release in the culture medium after treatment of HL60 cells with compound 1.
Table 2
treatment of HL60 cells with compound 1 was measured, using
doxorubicin as positive control (Table 1).
FACS analysis of HL60 cells treated with compound 1 for 24 h
SubG1
G0/G1
S phase
G2/M
After 24 h, caspase 3 activity was dose-dependently activated
by treatment with compound 1, with a 3-fold increase at 50 lM.
Thus compound 1 is able to stimulate apoptosis through the acti-
vation of caspase 3.
However the moderate activation of the caspase 3 cannot
account for the whole cytotoxic effect of compound 1. Therefore,
the release of cytosolic LDH into the culture medium as a marker
for cell necrosis was also measured (Fig. 4).
HL60 24 h
Doxo 50 nM
1.2
1.8
2.7
10.9
69.5
47.6
14.5
48.3
46.8
14.7
48.7
34.4
44.7
34.4
14.6
2.5
49.3
4.3
8.0
1.3
1 5
l
M
1 20
1 50
l
l
M
M
DNA was tagged with propidium iodide and results expressed as the percentage of
cells in the different phases of cell cycle.
After 24 h, the total LDH activity was not noticeably modified
indicating that the cell number was unaffected at that time.
However, compound 1 induced a clear release of LDH into the
by competing with ATP, in agreement with the in silico experi-
ments and chemical design work.
By contrast, compounds 3 and 4 did not show significant inhibi-
tion, while being structurally related. From these very informative
results, we tentatively established a preliminary structure–activity
culture medium at concentrations above 20 lM indicating that
cells entered the necrotic process. After 48 h, a dose-dependent
decrease of total LDH paralleled the cytotoxic effect of com-
pound 1 while the release of LDH remained observable. In the
same conditions, 1 lM doxorubicin elicited a reduction in the
relationship. The calculated Ki of 4.5 and 9 lM for compounds 1
total LDH content without any discernible leakage of the cyto-
solic LDH. Therefore we can conclude that necrosis is involved
in the cell death mode of compound 1. Finally, a possible effect
of compound 1 on cell division was investigated. As shown in
Table 2, doxorubicin promoted an early blockade of the cell cycle
in phase G2/M. Compound 1 did not block the cell cycle but pro-
moted a direct and dose-dependent accumulation of dead cells
(SubG1 phase). This is in line with the cytotoxic effect of com-
pound 1 in the non-dividing cell line EPC demonstrating that
compound 1 could promote cell death irrespective of the cell
cycle.
Guided by the information obtained from the initial virtual
screening, a series of original molecules was synthesized and eval-
uated for their effect on Pontin and potential anti-cancer activity.
Among the four selected candidates from the docking process, only
compounds 1 and 2 displayed a comparable effect on Pontin ATP-
ase activity. However, only compound 1 displayed a cytotoxic
effect, while compound 2 had no noticeable effect on cell growth.
This lack of activity of compound 2 could result from cell perme-
ability constraint, or instability. Further measures of the biological
activity of compound 1 emphasized the induction of apoptotic cell
death together with necrosis. This latter effect could be directly
related to the cellular balance of ATP regulation. With this new
lead in hand, we are working on the establishment of a structure
activity-relationship in order to find agents with lower IC50. Paral-
lel to this study, is the search for a deeper understanding of the
mechanism of action on the biological target.
and 2, respectively, suggested that the 3-hydroxy-5-phenyl-2-pyri-
done moiety was more suited for the inhibitory activity than the 3-
hydroxy-2-quinolone core. Moreover, the absence of inhibition
observed for compounds 3 and 4 in the enzymatic assay high-
lighted the crucial role of the
a
-aminoamide function (R1–NH–
CO–CH(NH2)–R2), characteristic of compounds 1 and 2, for the
interaction with the catalytic center.
Cell culture, cell proliferation assay, cell cycle analysis, flow
cytometric detection of apoptosis, caspase activity assay and
necrosis assay were performed as previously detailed.35 Only com-
pound 1 demonstrated an antiproliferative activity in cancer cell
lines at 10À5 M (Fig. 2). IC50 of 9 and 15
lM were measured respec-
tively for KB and HL60 cells after 72 h of exposure. The other syn-
thesized molecules displayed no cytotoxic activity, including
compound 2 despite its ability to partially inhibit Pontin ATPase
activity in vitro. Compound 1 also reduced cell numbers with an
IC50 of 25
effect.
lM in the non-dividing EPC cells, suggesting a cytotoxic
The mode of action of compound 1 in HL60 cells was then inves-
tigated in more detail. First, apoptosis was evaluated with annexin
and 7-AAD labeling using FACS analysis. A dose-dependent cell
apoptosis was evidenced in HL60, demonstrating an early cell
death after 24 h of exposure at a concentration of 20
l
M, increas-
ing at 48 h. At 50
24 h (Fig. 3).
lM, compound 1 induced 95% of cell death at
As it is generally assumed that caspase 3 is the terminal effector
in the apoptotic cascade pathway, the activation of caspase 3 after

2516
J. Elkaim et al. / Bioorg. Med. Chem. Lett. 24 (2014) 2512–2516
21. Izumi, N.; Yamashita, A.; Iwamatsu, A.; Kurata, R.; Nakamura, H.; Saari, B.;
Acknowledgments
Hirano, H.; Anderson, P.; Ohno, S. Sci. Signal. 2010, 3(ra2), 7.
22. Kim, S. J.; DeStefano, M. A.; Oh, W. J.; Wu, C. C.; Vega-Cotto, N. M.; Finlan, M.;
Liu, D.; Su, B.; Jacinto, E. Mol. Cell 2012, 48, 875.
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24. Dugan, K. A.; Wood, M. A.; Cole, M. D. Oncogene 2002, 21, 5835.
25. Grigoletto, A.; Neaud, V.; Allain-Courtois, N.; Lestienne, P.; Rosenbaum, J. Mol.
Cancer Res. 2013, 11, 133.
26. Elkaim, J.; Castroviejo, M.; Bennani, D.; Taouji, S.; Allain, N.; Laguerre, M.;
Rosenbaum, J.; Dessolin, J.; Lestienne, P. Biochem. J. 2012, 443, 549.
27. Lalut, J.; Tripoteau, L.; Marty, C.; Bares, H.; Bourgougnon, N.; Felpin, F.-X. Bioorg.
Med. Chem. Lett. 2012, 22, 7461.
This project was supported by grants from Equipe Labélisée
Ligue Contre le Cancer 2011, Institut National du Cancer grant
PLBIO10-155 and Grant ‘‘Institut National du Cancer – Direction
Générale de l’Offre de Soins – Institut National de la Santé et de
la Recherche Médicale 6046’’. The authors wish to thank the
Association pour la Recherche sur le Cancer, MENRT, CNRS and
Inserm for their support.
28. Commandeur, C.; Chalumeau, C.; Dessolin, J.; Laguerre, M. Eur. J. Org. Chem.
2007, 3045.
29. Trott, O.; Olson, A. J. J. Comput. Chem. 2010, 31, 455.
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34. 34(S)-1-(4-(4-Hydroxy-2-oxo-5-phenyl-1,2-dihydropyridin-3-yl)phenylamino)-3-
(1H-indol-3-yl)-1-oxopropan-2-aminium chloride 1: Trifluoroacetic acid (TFA,
500
l
L) was added dropwise to a stirred solution of 7a (100 mg, 0.13 mmol,
L) at 0 °C. The solution was stirred at 0 °C for 30 min
1 equiv) in CH2Cl2 (500
l
then concentrated under vacuum to provide the amine as a TFA salt. The
product was then diluted with EtOH and 1 M HCl and concentrated to provide
the chloride salt. The residue was diluted with EtOH (5 mL) then Pd/C
(10 mol %, 14 mg, 0.1 equiv) was added and the suspension was placed under
H2 atmosphere (balloon) and stirred at room temperature for 12 h. The
solution was diluted with EtOH (20 mL) and heated to 60 °C then warm filtered
over a celite pad. The cake was washed twice with warm EtOH. Combined
organic fractions were concentrated to provide the crude compound. The
product was purified by octadecyl-functionalized silica gel column
chromatography (100% H2O to 100% MeOH) to afford the chloride salt in 63%
yield as an amorphous white solid. 1H NMR (300 MHz, CD3OD) d 8.04 (1H, d,
J = 8.1 Hz), 7.69 (1H, d, J = 7.8 Hz), 7.60–7.00 (13H, m), 4.26 (1H, t, J = 7.0 Hz),
3.50 (1H, dd, J = 6.8 14.5 Hz), 3.37 (1H, dd, J = 6.8 14.5 Hz); 13C NMR (75 MHz,
CD3OD) d 167.6, 164.5, 160.3, 137.7, 136.87, 136.80, 131.6, 130.7, 129.2, 128.1,
126.9, 124.2, 123.3, 121.9, 120.4, 118.9, 117.8, 116.4, 115.0, 111.26, 111.20,
106.6, 54.3, 27.7; IR (ZnSe)
m 2967, 2902, 1706, 1614, 1575, 1471, 1410, 1401,
1375, 1336, 1211, 1057, 751, 701; HRMS (ESI): m/z calcd for
[(M+H)+] = 465.1927, found = 465.1920.
(S)-1-(4-(4-Hydroxy-2-oxo-1,2-dihydroquinolin-3-yl)phenylamino)-3-(1H-indol-
3-yl)-1-oxopropan-2-aminium chloride 2: Following the procedure described for
the synthesis of 1, the compound was obtained from 7b in 57% yield as an
amorphous solid after purification by octadecyl-functionalized silica gel
column chromatography (100% H2O to 100% MeOH). 1H NMR (300 MHz,
CD3OD) d 7.69 (1H, d, J = 7.5 Hz), 7.57 (1H, d, J = 8.4 Hz), 7.51–7.30 (7H, m),
7.29–7.00 (3H, m), 4.26 (1H, t, J = 6.9 Hz), 3.50 (1H, dd, J = 6.6, 14.7 Hz), 3.37
(1H, dd, J = 7.5, 14.4 Hz); 13C NMR (75 MHz, CD3OD) d 167.2, 162.5, 159.5,
137.0, 136.9, 134.1, 132.9, 131.4, 129.1, 128.8, 128.1, 127.3, 126.9, 124.3, 121.5,
120.4, 118.9, 117.8, 116.6, 111.3, 111.2, 106.5, 54.3, 27.5; IR (ZnSe)
m 3250,
3028, 2966, 2921, 1674, 1634, 1600, 1517, 1378, 1202, 1142, 1045, 834, 758,
675 cmÀ1; HRMS (ESI): m/z calcd for [(M+H)+]: 439.1765, found: 439.1801.
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